Over the past decade, numerous methods that combine the power of combinatorial chemistry and high-throughput screening have been developed to generate novel ligands that are critical to both basic research and therapeutic applications. Among these methods, the adaptive molecular evolution techniques with RNA, also known as SELEX (Tuerk and Gold, “Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase,” Science 249:505-510 (1990)), is particularly powerful thanks to the unique features of the RNA molecules, which not only carry information for their own replication but also fold into well-defined shapes. The novel ligands, or aptamers (Ellington and Szostak, “In vitro Selection of RNA Molecules that Bind Specific Ligands,” Nature 346:818-822 (1990)), generated by this SELEX process are capable of binding to a wide variety of targets with high affinity and specificity (Gold et al, “Diversity of Oligonucleotide Functions,” Ann. Rev. Biochem. 64:763-797 (1995); Wilson and Szostak, “In vitro Selection of Functional Nucleic Acids,” Ann. Rev. Biochem. 68:611-647 (1999)).
There has been a growing need in the drug discovery and functional genomics fields to develop methods that would yield aptamers against multiple targets in a single selection experiment. Experiments have been performed against multi-subunit enzymes (Brown and Gold, “Template Recognition by an RNA-dependent RNA Polymerase: Identification and Characterization of Two RNA Binding Sites on Qβ Replicase,” Biochemistry 34:14765-14774 (1995); Brown and Gold, “RNA Replication by Qβ Replicase: a Working Model,” Proc. Natl. Acad. Sci. USA 93:11558-11562 (1996)), viral particles (Pan et al, “Isolation of Virus-neutralizing RNAs from a Large Pool of Random Sequences,” Proc. Natl. Acad. Sci. USA 92:11509-11513 (1995)), organelles (Ringquist et al, “High-affinity RNA Ligands to Escherichia coli Ribosomes and Ribosomal Protein SI: Comparison of Natural and Unnatural Binding Sites,” Biochemistry 34:3640-3648 (1995)), and entire cells (Morris et al, “High Affinity Ligands from in vitro Selection: Complex Targets,” Proc. Natl. Acad. Sci. USA 95:2902-2907 (1998)), with different degrees of success. In some cases, different families of ligands were identified for different targets in the mixture. However, these aptamers recognize the most abundant or easily recognizable target sites, which are not necessarily the most desired ones. No existing literature, i.e. none of the experiments performed so far and none of the currently available methods, offers an approach which is capable of generating different ligands to all of the targets in a mixture.
The most important step in the SELEX procedure is the partitioning step in which the target-binding species is physically separated from the non-binding species. The immobilization matrix used as partitioning device can act as an unwanted target to generate unwanted aptamers that often dominate the selected pool. Several methods are commonly in use to avoid this problem (Conrad et al, “In vitro Selection of Nucleic Acid Aptamers that Bind Proteins,” Methods in Enzymology 267:336-367 (1996)). First, matrix-binding species may be eliminated by negative selection against the matrix, i.e., collecting the fraction not bound to the matrix. However, in early cycles, when the copy number of each clone is low, this extra handling may increase the chance of stochastic events in which a particular sequence is lost. In later rounds, when the matrix bound species becomes the dominant sequence population in a pool, this method may not be efficient enough to eliminate them. Second, since the number of binding site on the surface of the matrix is fixed, increasing the amount of the target may change the ratio of the matrix-binding species to target-binding aptamers in favor of the recovery of the target-binding aptamers in the partitioning step. But the number of binding sites on the matrix can be extremely large compared to that on the target even at its highest level, thus rendering this method ineffective. In addition, higher target amounts will also favor the isolation of ligands with lower affinity, thus decreasing the efficiency of selection. Third, alternating use of different types of matrices should theoretically eliminate molecules binding to either. This method is less effective than might be expected because the difference between the commonly used matrices is not sufficient to discriminate against common aptamers that can bind via less specific hydrophobic interactions. Because of the lack of an efficient and specific negative selection method, the background problem is still a major reason of failure during in vitro selection experiments.